Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The platelet glycoprotein Ib/IX (GPIb/IX) receptor for von Willebrand factor (vWf) is believed to consist of a 1:1 heterodimeric complex (Du et al. 1987) between GPIb (160 kDa) and GPIX (17 kDa) in a noncovalent association. GPIb in turn consists of a disulfide-linked 140 kDa alpha chain (GPIb alpha) and a 22 kDa beta chain (GPIb beta) (Fitzgerald and Phillips 1989).
The GPIb/IX complex comprises one of the major transmembrane receptor complexes on blood platelets (Roth 1991; Lopez 1994; Clemetson and Clemetson 1995), mediating von Willebrand factor (vWF)-dependent platelet adhesion. The human autosomal dominant bleeding disorder termed platelet-type von Willebrand disease (PT-vWD) represents a naturally occurring model of an up-regulated GPIb/IX receptor (Miller and Castella 1982; Miller et al. 1983). In this disorder, abnormally low concentrations of the chemical modulator ristocetin are able to promote the interaction of vWF with GPIb/IX. Additionally, the platelets from such patients are aggregated at a lower shear force than required for normal platelets (Murata et al. 1993). One kindred of PT-vWD patients was found to have a single point mutation leading to a substitution of valine for glycine at residue 233 of the GPIb alpha chain (Miller et al. 1991). A second point mutation in very close proximity (substitution of valine for methionine at residue 239 (Russell and Roth 1993; Takahashi et al 1995) has been described in two additional kindreds displaying the PT-vWD phenotype (Weiss et al. 1982; Takahashi 1980).
In the 1980's, Miller et al. developed a series of monoclonal antibodies (mab) directed against the GP Ib/IX complex receptor for vWf. In particular, monoclonal antibody C-34 was characterized in detail and it was determined that mab C-34 recognized an epitope within the platelet glycoprotein Ib/IX complex (Miller et al. 1990). In this and subsequent work, Miller et al. showed that monoclonal antibodies C-34, AS-2 and AS-7 were potent inhibitors of the ristocetin-induced aggregation of normal platelets that was dependent upon von Willebrand factor. Miller et al. also showed that the epitopes for all three monoclonal antibodies lay within the GPIb/IX complex. Miller et al. were able to localize monoclonal antibody binding sites for AS-2 and AS-7 to the amino-terminal 45 kDa of GPIb alpha. The epitope for C-34 was recently localized to the extracellular portion of the GPIb alpha chain expressed on the surface of Chinese Hamster Ovary cells (Chambers et al. 1995). The failure of C-34 to bind to denatured GPIb alpha in Western blots (Ward and Berndt 1995; Clemetson and Hugli 1995), or to immunoprecipitate the extracellular region of GPIb alpha removed from platelets under a variety of experimental conditions (Miller et al. 1990) strongly suggests that the epitope recognized by C-34 is highly conformation-dependent. Recently Ward and Berndt have, however, now reported the successful immunoprecipitation by C-34 of a 1.His-Arg.293 amino-terminal fragment of 125I-labeled glycocalicin following digestion of the purified molecule by trypsin (Ward and Berndt 1995).
Attempts to define the binding sites for various monoclonal antibodies have led to the development of epitope libraries. Parmley and Smith developed a bacteriophage expression vector that could display foreign epitopes on its surface (Parmley and Smith 1988). This vector could be used to construct large collections of bacteriophage which could include virtually all possible sequences of a short (e.g. six-amino-acid) peptide. They also developed biopanning, which is a method for affinity-purifying phage displaying foreign epitopes using a specific antibody (see Parmley and Smith 1988; Cwirla et al. 1990; Scott and Smith 1990; Christian et al. 1992; Smith and Scott 1993).
After the development of epitope libraries, Smith et al. then suggested that it should be possible to use the bacteriophage expression vector and biopanning technique of Parmley and Smith to identify epitopes from all possible sequences of a given length. This led to the idea of identifying peptide ligands for antibodies by biopanning epitope libraries, which could then be used in vaccine design, epitope mapping, the identification of genes, and many other applications (Parmley and Smith 1988; Scott 1992).
Using epitope libraries and biopanning, researchers searching for epitope sequences found instead peptide sequences which mimicked the epitope, i.e., sequences which did not identify a continuous linear native sequence or necessarily occur at all within a natural protein sequence. These mimicking peptides are called mimotopes. In this manner, mimotopes of various binding sites/proteins have been found. LaRocca et al. (1992) expressed a mimotope of the human breast epithelial mucin tandem repeat in Escherichia coli. Balass et al. (1993) identified a hexapeptide that mimics a conformation-dependent binding site of the acetylcholine receptor. Hobart et al. (1993) isolated a mimotope that mimics the C6 epitope (the epitope for the sixth component of complement).
The sequences of these mimotopes, by definition, do not identify a continuous linear native sequence or necessarily occur in any way in a naturally-occurring molecule, i.e. a naturally occuring protein. The sequences of the mimotopes merely form a peptide which functionally mimics a binding site on a naturally-occurring protein. For example, the mimotope of Balass et al. (1993) mimics the binding site of the acetylcholine receptor.
Many of these mimotopes are short peptides. The availability of short peptides which can be readily synthesized in large amounts and which can mimic naturally-occurring sequences (i.e. binding sites) offers great potential application.
A need continues to exist, therefore, for the elucidation of useful mimotopes.